Biomedical subjects

S Cusack

Publications and source records attributed to S Cusack.

At least 19 recordsLinked to original sources

PubMed2006

A comparison of paediatric soccer, gaelic football and rugby injuries presenting to an emergency department in Ireland.

OBJECTIVES: Children presenting with sport related injuries (SRIs) as a result of soccer, rugby and gaelic football are frequently seen in an emergency medicine (EM) setting in Ireland. A comparison of the demographics of injuries in these three sports has however not previously been performed. The purpose of this study was to provide up-to-date data on the nature of these SRIs. METHOD: Data was collected retrospectively on all children (<17 years of age), injured in these three sports, presenting to an emergency medicine department over 6 months, and was entered into a database for analysis. RESULTS: Retrospective analysis was performed on 23,000 charts, and 409 SRIs were identified over a 6-month period. None of the children reported using any form of protective gear, and 27% reported a previous presentation to the emergency department with a SRI. Most injuries were as a result of soccer (56%), with 24% occurring in gaelic football, and 20% occurring in rugby. The predominant mechanism of injury was different in each sport, in soccer-falls (38%), in gaelic football-collisions with objects (balls) (37%), and in rugby-collision with persons (55%). Although the predominant type of injury in soccer and gaelic football was a fracture, accounting for 50% and 42% of injuries, respectively, in rugby however, skin/soft tissue injuries presented more commonly, accounting for 44% of injuries. When the general site of injury was investigated, the upper limb accounted for the majority of SRIs in each sport. In the management of SRIs, oral analgesics were prescribed in 50%, however, it was observed that no use was made of topical, intramuscular or rectal analgesic routes of administration. In addition it was observed that RICE/general injury advice was given in only 27%, physiotherapy was requested in 2%, and no injury prevention advice was given to any child. Overall, 8% required admission. CONCLUSIONS: The data provided from this study may raise awareness of the nature of SRIs affecting children in each of these three sports, and may be useful in formulating much needed injury prevention strategies.

Adolescent↗

PubMed2006

First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value.

The EC 'Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, approximately 170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.

Animals↗

PubMed2006

Automation of sample mounting for macromolecular crystallography.

A standard sample holder and vial for cryocooled macromolecular crystals has been defined for use with robotic sample changers. This SPINE standard sample holder is a modified version, with added features and specifications, of sample holders in common use. In particular, the SPINE standard meets the precision required for automatic sample exchange and includes a cap that is identified by a two-dimensional datamatrix code as well as an optional vial. At the ESRF, the sample holder standard is in use with the EMBL/ESRF/BM14 robotic sample changer (SC3) which is installed on eight beamlines. The SC3 can hold up to 50 crystals stored in five baskets. A datamatrix reader in the SC3 ensures safe management of the sample flow and facilitates fully automatic screening and characterization of samples. Tools for handling and transporting 50 samples in a dry shipping dewar have been developed. In addition to the SC3, the SPINE sample holder is currently compatible with a number of other robotic sample changers.

Automation↗

PubMed2006

High-throughput sample handling and data collection at synchrotrons: embedding the ESRF into the high-throughput gene-to-structure pipeline.

An automatic data-collection system has been implemented and installed on seven insertion-device beamlines and a bending-magnet beamline at the ESRF (European Synchrotron Radiation Facility) as part of the SPINE (Structural Proteomics In Europe) development of an automated structure-determination pipeline. The system allows remote interaction with beamline-control systems and automatic sample mounting, alignment, characterization, data collection and processing. Reports of all actions taken are available for inspection via database modules and web services.

Data Collection↗

PubMed2005

The effect of conjugated linoleic acid on the viability and metabolism of human osteoblast-like cells.

Studies in experimental animals and murine osteoblast cells in culture have produced conflicting findings on the effect of conjugated linoleic acid (CLA) on bone formation. The present study investigated the influence of CLA on viability and metabolism of two human osteoblast-like cell lines (SaOS2 and MG63). Both cell lines were exposed to increasing concentrations (0-50 microM) of CLA either as pure cis (c) 9: trans (t) 11 and t10:c12 CLA isomers or a blend of isomers, or linoleic acid (C18:2). Cell cytotoxicity and degree of DNA fragmentation were unaffected by any fatty acid treatment. PGE2 biosynthesis by both cell lines was variably reduced by CLA isomer blend and t10:c12 CLA, but not c9:t11 CLA. Alkaline phosphatase activity was variably increased by all CLA treatments. These results suggest a lack of cytotoxic effect of CLA on human osteoblast-like cells and tentatively suggest a possible beneficial effect on bone formation in humans.

Alkaline Phosphatase↗

PubMed2005

The effect of conjugated linoleic acid on transepithelial calcium transport and mediators of paracellular permeability in human intestinal-like Caco-2 cells.

Conjugated linoleic acid (CLA) increases paracellular permeability across human intestinal-like Caco-2 cell monolayers, which transport Ca predominantly by the transcellular route. In vivo, however, paracellular Ca transport is the predominant route of Ca transport. Therefore, the objective of this study was to investigate the effect of CLA on transepithelial Ca transport in Caco-2 cells transporting Ca predominantly by the paracellular route. Cells were seeded onto permeable transport membranes and allowed to differentiate, over 14 d, into intestinal-like cell monolayers. Monolayers (n=9/treatment) were exposed to 0 (control) or 80 microM- 18:2, -cis-9, trans-11 CLA or -trans-10, cis-12 CLA for 14 d prior to Ca transport studies. Overall transepithelial Ca transport as well as transcellular and parcellular Ca transport was significantly increased (P<0.001) by exposure of Caco-2 cells to both isomers of CLA, an effect which appeared to be related to altered localization of zona occludens 1 (a tight junction protein).

Biological Transport↗

PubMed2004

Ladder fall injuries: patterns and cost of morbidity.

Ladder fall injuries are associated with a wide spectrum of injury patterns. However, the healthcare cost of these injuries is unknown. The aim of this study was to determine the healthcare cost and duration of the morbidity associated with ladder fall injuries. A retrospective observational study involving patients with ladder injuries who presented to a Level 1 Trauma Centre over a 3-year period was performed. Patients then underwent a structured telephone interview to provide information about the duration of their disability and unemployment. There were 72 patients in the study, of whom 89% were male. Thirteen patients (18%) required hospital admission. The median length and cost of hospital stay was 1 week and 3555 (US$ 3950), respectively. The median duration of disability and unemployment was 6 weeks. The longest duration of disability was associated with foot fractures. The majority of patients fell between 5 and 10ft, and fell at work. The upper extremity was the most commonly injured anatomical region. There were no fatalities. Ladder injury falls are expensive to the health service and may warrant investment in their prevention.

Accidental Falls↗

PubMed2001

Crystal structure of an early protein-RNA assembly complex of the signal recognition particle.

The signal recognition particle (SRP) is a universally conserved ribonucleoprotein complex that mediates the cotranslational targeting of secretory and membrane proteins to cellular membranes. A crucial early step in SRP assembly in archaea and eukarya is the binding of protein SRP19 to specific sites on SRP RNA. Here we report the 1.8 angstrom resolution crystal structure of human SRP19 in complex with its primary binding site on helix 6 of SRP RNA, which consists of a stem-loop structure closed by an unusual GGAG tetraloop. Protein-RNA interactions are mediated by the specific recognition of a widened major groove and the tetraloop without any direct protein-base contacts and include a complex network of highly ordered water molecules. A model of the assembly of the SRP core comprising SRP19, SRP54, and SRP RNA based on crystallographic and biochemical data is proposed.

Amino Acid Sequence↗

PubMed2001

Structure of the fiber head of Ad3, a non-CAR-binding serotype of adenovirus.

Adenoviruses of serotype Ad3 (subgenus B) use a still-unknown host cell receptor for viral attachment, whereas viruses from all other known subgenera use the coxsackie and adenovirus receptor (CAR). The receptor binding domain (head) of the Ad3 fiber protein has been expressed in Escherichia coli inclusion bodies. After denaturation and renaturation using a rapid dilution method, crystals of trimeric head were obtained. The 1.6 A resolution X-ray structure shows a strict conservation of the beta-sheet scaffold of the protein very similar to the head structures of the CAR-binding serotypes Ad2, Ad5, and Ad12. The conformation of the loops is different, with the exception of the AB loop, which forms the center of the interface in the Ad12-CAR complex structure. The structure explains why a mutation in Ad5 of one residue in the AB loop to glutamic acid, as in Ad3, abrogates binding to CAR. It is possible that the Ad3 receptor binding site is nevertheless situated similar to the CAR binding site, although it cannot be excluded that other regions of the relatively hydrophobic head surface may be used.

Adenoviruses, Human↗

PubMed2001

A succession of substrate induced conformational changes ensures the amino acid specificity of Thermus thermophilus prolyl-tRNA synthetase: comparison with histidyl-tRNA synthetase.

We describe the recognition by Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) of proline, ATP and prolyl-adenylate and the sequential conformational changes occurring when the substrates bind and the activated intermediate is formed. Proline and ATP binding cause respectively conformational changes in the proline binding loop and motif 2 loop. However formation of the activated intermediate is necessary for the final conformational ordering of a ten residue peptide ("ordering loop") close to the active site which would appear to be essential for functional tRNA 3' end binding. These induced fit conformational changes ensure that the enzyme is highly specific for proline activation and aminoacylation. We also present new structures of apo and AMP bound histidyl-tRNA synthetase (HisRS) from T. thermophilus which we compare to our previous structures of the histidine and histidyl-adenylate bound enzyme. Qualitatively, similar results to those observed with T. thermophilus prolyl-tRNA synthetase are found. However histidine binding is sufficient to induce the co-operative ordering of the topologically equivalent histidine binding loop and ordering loop. These two examples contrast with most other class II aminoacyl-tRNA synthetases whose pocket for the cognate amino acid side-chain is largely preformed. T. thermophilus prolyl-tRNA synthetase appears to be the second class II aminoacyl-tRNA synthetase, after HisRS, to use a positively charged amino acid instead of a divalent cation to catalyse the amino acid activation reaction.

Acylation↗

PubMed2001

Crystal structure of the human nuclear cap binding complex.

The heterodimeric nuclear cap binding complex (CBC) binds to 5'-capped polymerase II transcripts. It enhances the efficiency of several mRNA maturation steps and is essential for U snRNA nuclear export in multicellular eukaryotes. The 2A crystal structure of human CBC shows that the large subunit, CBP80, comprises three domains, each containing consecutive helical hairpins and resembling the so-called MIF4G domain found in several other proteins involved in RNA metabolism. The small subunit, CPB20, has an RNP fold and associates with the second and third domains of CBP80. Site-directed mutagenesis revealed 4 residues of CBP20 which are critical for cap binding. A model for cap binding is proposed based on these results and the known mode of binding of RNA to RNP domains.

Amino Acid Sequence↗

PubMed2001

Hierarchical assembly of the Alu domain of the mammalian signal recognition particle.

The mammalian signal recognition particle (SRP) catalytically promotes cotranslational translocation of signal sequence containing proteins across the endoplasmic reticulum membrane. While the S-domain of SRP binds the N-terminal signal sequence on the nascent polypeptide, the Alu domain of SRP temporarily interferes with the ribosomal elongation cycle until the translocation pore in the membrane is correctly engaged. Here we present biochemical and biophysical evidence for a hierarchical assembly pathway of the SRP Alu domain. The proteins SRP9 and SRP14 first heterodimerize and then initially bind to the Alu RNA 5' domain. This creates the binding site for the Alu RNA 3' domain. Alu RNA then undergoes a large conformational change with the flexibly linked 3' domain folding back by 180 degrees onto the 5' domain complex to form the final compact Alu ribonucleoprotein particle (Alu RNP). We discuss the possible mechanistic consequences of the likely reversibility of this final step with reference to translational regulation by the SRP Alu domain and with reference to the structurally similar Alu RNP retroposition intermediates derived from Alu elements in genomic DNA.

Alu Elements↗

PubMed2001

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism.

Targeting of adenovirus (Ad)-encoded therapeutic genes to specific cell types has become a major goal in gene therapy. Redirecting the specificity of infection requires the abrogation of the natural interaction between the viral fiber and its cellular receptors (CAR) and the simultaneous introduction of a new binding specificity into the viral capsid. To abrogate the natural affinity of the fiber, we have mutated residues presumed to be directly or indirectly involved in CAR-binding in the knob domain of the fiber protein. These residues are located in the AB loop (Ser408) and in the DG loop (Tyr491, Ala494, Ala503). The mutations Ser408Glu, Tyr491Asp, Ala494Asp and Ala503Asp did not prevent the incorporation of trimeric fibers in the viral capsid but led to loss of CAR binding in vitro. Infectivity of the mutant viruses could be restored in vitro by introducing a ligand at the C-terminal end of the knob, confirming that the reduced infectivity of the fiber-modified virus was due to an impaired interaction of the viral particle with the CAR receptor. However, after systemic delivery, the in vivo biodistribution of impaired CAR-binding viruses without addition of a specific ligand was not altered when compared with wild-type Ad.

Adenoviruses, Human↗

PubMed2001

Epidemic dystonia in Cork.

We describe a case series of five patients with unusual presentations of acute dystonia seen over a 2-week period. Haloperidol, prescribed and allegedly purchased 'on the street', was thought to be a possible cause in each case. Bizarre clinical presentations and delayed onset of symptoms may make diagnosis difficult. A high index of suspicion combined with a careful drug history is essential.

Adolescent↗

PubMed2000

Kinetic analysis of adenovirus fiber binding to its receptor reveals an avidity mechanism for trimeric receptor-ligand interactions.

Most adenoviruses bind to the N-terminal immunoglobulin domain D1 of the coxsackievirus and adenovirus receptor via the head part of their fiber proteins. Three receptor molecules can bind per fiber head. We expressed the D1 domain and the adenovirus type 2 fiber head in bacteria and studied binding interactions by surface plasmon resonance measurements. When receptor domains bind adenovirus fiber independently of each other, the dissociation constant is 20-25 nm. However, when adenovirus fiber binds to receptors immobilized on the sensor chip, a situation better mimicking adenovirus binding to receptors on the cell surface, the dissociation constant was around 1 nm. Kinetic analysis shows that this happens via an avidity mechanism; three identical interactions with high on and off rate constants lead to tight binding of one fiber head to three receptor molecules with a very low overall off rate. The avidity mechanism could be used by other viruses that have multimeric adhesion proteins to attach to target cells. It could also be more general to trimeric receptor-ligand interactions, including those involved in intracellular signaling.

Adenoviridae↗

PubMed2000

Dimeric structure of the coxsackievirus and adenovirus receptor D1 domain at 1.7 A resolution.

BACKGROUND: The coxsackievirus and adenovirus receptor (CAR) comprises two extracellular immunoglobulin domains, a transmembrane helix and a C-terminal intracellular domain. The amino-terminal immunoglobulin domain (D1) of CAR is necessary and sufficient for adenovirus binding, whereas the site of coxsackievirus attachment has not yet been localized. The normal cellular role of CAR is currently unknown, although CAR was recently proposed to function as a homophilic cell adhesion molecule. RESULTS: The human CAR D1 domain was bacterially expressed and crystallized. The structure was solved by molecular replacement using the structure of CAR D1 bound to the adenovirus type 12 fiber head and refined to 1.7 A resolution, including individual anisotropic temperature factors. The two CAR D1 structures are virtually identical, apart from the BC, C"D, and FG loops that are involved both in fiber head binding and homodimerization in the crystal. Analytical equilibrium ultracentrifugation shows that a dimer also exists in solution, with a dissociation constant of 16 microM. CONCLUSIONS: The CAR D1 domain forms homodimers in the crystal using the same GFCC'C" surface that interacts with the adenovirus fiber head. The homodimer is very similar to the CD2 D1-CD58 D1 heterodimer. CAR D1 also forms dimers in solution with a dissociation constant typical of other cell adhesion complexes. These results are consistent with reports that CAR may function physiologically as a homophilic cell adhesion molecule in the developing mouse brain. Adenovirus may thus have recruited an existing and conserved interaction surface of CAR to use for its own cell attachment.

Adenoviruses, Human↗

PubMed2000

Structure and assembly of the Alu domain of the mammalian signal recognition particle.

The Alu domain of the mammalian signal recognition particle (SRP) comprises the heterodimer of proteins SRP9 and SRP14 bound to the 5' and 3' terminal sequences of SRP RNA. It retards the ribosomal elongation of signal-peptide-containing proteins before their engagement with the translocation machinery in the endoplasmic reticulum. Here we report two crystal structures of the heterodimer SRP9/14 bound either to the 5' domain or to a construct containing both 5' and 3' domains. We present a model of the complete Alu domain that is consistent with extensive biochemical data. SRP9/14 binds strongly to the conserved core of the 5' domain, which forms a U-turn connecting two helical stacks. Reversible docking of the more weakly bound 3' domain might be functionally important in the mechanism of translational regulation. The Alu domain structure is probably conserved in other cytoplasmic ribonucleoprotein particles and retroposition intermediates containing SRP9/14-bound RNAs transcribed from Alu repeats or related elements in genomic DNA.

Alu Elements↗

PubMed2000

Mutual conformational adaptations in antigen and antibody upon complex formation between an Fab and HIV-1 capsid protein p24.

BACKGROUND: Elucidating the structural basis of antigen-antibody recognition ideally requires a structural comparison of free and complexed components. To this end we have studied a mouse monoclonal antibody, denoted 13B5, raised against p24, the capsid protein of HIV-1. We have previously described the first crystal structure of intact p24 as visualized in the Fab13B5-p24 complex. Here we report the structure of the uncomplexed Fab13B5 at 1.8 A resolution and analyze the Fab-p24 interface and the conformational changes occurring upon complex formation. RESULTS: Fab13B5 recognizes a nearly continuous epitope comprising a helix-turn-helix motif in the C-terminal domain of p24. Only 4 complementarity-determining regions (CDRs) are in contact with p24 with most interactions being by the heavy chain. Comparison of the free and complexed Fab reveals that structural changes upon binding are localized to a few side chains of CDR-H1 and -H2 but involve a larger, concerted displacement of CDR-H3. Antigen binding is also associated with an 8 degrees relative rotation of the heavy and light chain variable regions. In p24, small conformational changes localized to the turn between the two helices comprising the epitope result from Fab binding. CONCLUSIONS: The relatively small area of contact between Fab13B5 and p24 may be related to the fact that the epitope is a continuous peptide rather than a more complex protein surface and correlates with a relatively low affinity of antigen and antibody. Despite this, a significant quaternary structural change occurs in the Fab upon complex formation, with additional smaller adaptations of both antigen and antibody.

Amino Acid Sequence↗